Substituted Indolyl-Alkyl-Amino-Derivatives as Inhibitors of Histone Deacetylase

ABSTRACT

This invention comprises the novel compounds of formula (I) wherein R 1 , R 2 , R 3 , R 4 , R 5 , X and Y have defined meanings, having histone deacetylase inhibiting enzymatic activity; their preparation, compositions containing them and their use as a medicine.

This invention concerns compounds having histone deacetylase (HDAC)inhibiting enzymatic activity. It further relates to processes for theirpreparation, to compositions comprising them, as well as their use, bothin vitro and in vivo, to inhibit HDAC and as a medicine, for instance asa medicine to inhibit proliferative conditions, such as cancer andpsoriasis.

Nuclear histones are known as integral and dynamic components of themachinery responsible for regulating gene transcription and otherDNA-templated processes such as replication, repair, recombination, andchromosome segregation. They are the subject of post-translationalmodifications including acetylation, phosphorylation, methylation,ubiquitination, and ADP-ribosylation.

Histone deacetylase(s), herein referred to as “HDACs”, are enzymes thatcatalyze the removal of the acetyl modification on lysine residues ofproteins, including the core nucleosomal histones H2A, H2B, H3 and H4.Together with histone acetyltransferase(s), herein referred to as“HATs”, HDACs regulate the level of acetylation of the histones. Thebalance of acetylation of nucleosomal histones plays an important rolein transcription of many genes. Hypoacetylation of histones isassociated with condensed chromatin structure resulting in therepression of gene transcription, whereas acetylated histones areassociated with a more open chromatin structure and activation oftranscription.

Eleven structurally related HDACs have been described and fall into twoclasses. Class I HDACs consist of HDAC 1, 2, 3, 8 and 11 whereas classII HDACs consist of HDAC 4, 5, 6, 7, 9 and 10. Members of a third classof HDACs are structurally unrelated to the class I and class II HDACs.Class I/II HDACs operate by zinc-dependent mechanisms, whereas class IIIHDACs are NAD-dependent.

In addition to histones, other proteins have also been the substrate foracetylation, in particular transcription factors such as p53, GATA-1 andE2F; nuclear receptors such as the glucocorticoid receptor, the thyroidreceptors, the estrogen receptors; and cell-cycle regulating proteinssuch as pRb. Acetylation of proteins has been linked with proteinstabilization, such as p53 stabilization, recruitment of cofactors andincreased DNA binding. p53 is a tumour suppressor that can induce cellcycle arrest or apoptosis in response to a variety of stress signals,such as DNA damage. The main target for p53-induced cell cycle arrestseems to be the p21 gene. Next to its activation by p53, p21 has beenidentified by virtue of its association with cyclin/cyclin-dependentkinase complexes resulting in cell cycle arrest at both G1 and G2phases, its up-regulation during senescence, and its interaction withthe proliferating cell nuclear antigen.

The study of inhibitors of HDACs indicates that they play an importantrole in cell cycle arrest, cellular differentiation, apoptosis andreversal of transformed phenotypes.

The inhibitor Trichostatin A (TSA), for example, causes cell cyclearrest at both G1 and G2 phases, reverts the transformed phenotype ofdifferent cell lines, and induces differentiation of Friend leukemiacells and others. TSA (and suberoylanilide hydroxamic acid SAHA) havebeen reported to inhibit cell growth, induce terminal differentiation,and prevent the formation of tumours in mice (Finnin et al., Nature,401: 188-193, 1999).

Trichostatin A has also been reported to be useful in the treatment offibrosis, e.g. liver fibrosis and liver chirrhosis. (Geerts et al.,European Patent Application EP 0 827 742, published 11 Mar. 1998).

The pharmacophore for HDAC inhibitors consists of a metal-bindingdomain, which interacts with the zinc-containing active site of HDACs, alinker domain, and a surface recognition domain or capping region, whichinteracts with residues on the rim of the active site.

Inhibitors of HDACs have also been reported to induce p21 geneexpression. The transcriptional activation of the p21 gene by theseinhibitors is promoted by chromatin remodelling, following acetylationof histones H3 and H4 in the p21 promoter region. This activation of p21occurs in a p53-independent fashion and thus HDAC inhibitors areoperative in cells with mutated p53 genes, a hallmark of numeroustumours.

In addition HDAC inhibitors can have indirect activities such asaugmentation of the host immune response and inhibition of tumourangiogenesis and thus can suppress the growth of primary tumours andimpede metastasis (Mai et al., Medicinal Research Reviews, 25: 261-309,2005).

In view of the above, HDAC inhibitors can have great potential in thetreatment of cell proliferative diseases or conditions, includingtumours with mutated p53 genes.

Patent application EP1472216 published on Aug. 14, 2003 disclosesbicyclic hydroxamates as inhibitors of histone deacetylase.

Patent applications EP1485099, EP1485348, EP1485353, EP1485354,EP1485364, EP1485365, EP1485370, EP1485378, published on 18 Sep. 2003,amongst others, disclose substituted piperazinylpyrimidinylhydroxamicacids as inhibitors of histone deacetylase furthermore EP1485365discloses R306465.

Patent application EP1492534 published on 9 Oct. 2003, disclosescarbamic acid compounds comprising a piperazine linkage, as HDACinhibitors.

Patent application EP1495002 published on 23 Oct. 2003, disclosesubstituted piperazinyl phenyl benzamide compounds, as histonedeacetylase inhibitors.

Patent application EP1501508 published on 13 Nov. 2003, disclosesbenzamides as histone deacetylase inhibitors.

Patent application WO04/009536 published on 29 Jan. 2004, disclosesderivatives containing an alkyl linker between the aryl group and thehydroxamate, as histone deacetylase inhibitors.

Patent application EP1525199 published on 12 Feb. 2004, discloses(hetero)arylalkenyl substituted bicyclic hydroxamates, as histonedeacetylase inhibitors.

Patent application EP1572626 published on 24 Jun. 2004, disclosesarylene-carboxylic acid (2-amino-phenyl)-amide derivatives aspharmacological agents.

Patent application EP1581484 published on 29 Jul. 2004, disclosesderivatives of N-hydroxy-benzamide derivatives with anti-inflammatoryand antitumour activity.

Patent application EP1585735 published on 29 Jul. 2004, disclosessubstituted aryl hydroxamate derivatives as histone deacetylaseinhibitors.

Patent application EP1592667 published on 19 Aug. 2004, disclosesmono-acylated O-phenylenediamines derivatives as pharmacological agents.

Patent application EP1590340 published on 19 Aug. 2004, disclosesdiaminophenylene derivatives as histone deacetylase inhibitors.

Patent application EP1592665 published on 26 Aug. 2004, disclosesbenzamide derivatives as histone deacetylase inhibitors.

Patent application WO04/072047 published on 26 Aug. 2004, disclosesindoles, benzimidazoles and naphhimidazoles as histone deacetylaseinhibitors.

Patent application EP1608628 published on 30 Sep. 2004, discloseshydroxamates linked to non-aromatic heterocyclic ring systems as histonedeacetylase inhibitors.

Patent application EP1613622 published on 14 Oct. 2004, discloses oximederivatives as histone deacetylase inhibitors.

Patent application EP1611088 published on 28 Oct. 2004, discloseshydroxamate derivatives as histone deacetylase inhibitors.

Patent application WO05/028447 published on 31 Mar. 2005, disclosesbenzimidazoles as histone deacetylase inhibitors.

Patent applications WO05/030704 and WO05/030705 published on 7 Apr.2005, discloses benzamides as histone deacetylase inhibitors.

Patent application WO05/040101 published on 6 May 2005, disclosesacylurea connected and sulfonylurea connected hydroxamates as histonedeacetylase inhibitors.

Patent application WO05/040161 also published on 6 May 2005, disclosesbiaryl linked hydroxamates as histone deacetylase inhibitors.

Patent application WO05/075469 published on 18 Aug. 2005, disclosesthiazolyl hydroxamic acids and Thiadiazolyl hydroxamic acids as histonedeacetylase inhibitors.

Patent application WO05/086898 published on 22 Sep. 2005, disclosesheteropentacyclic hydroxamic acids as histone deacetylase inhibitors.

Patent application WO05/092899 published on 6 Oct. 2005, disclosesalkenylbenzamides as histone deacetylases.

The compounds of the present invention differ from the prior art instructure, in their pharmacological activity and/or pharmacologicalpotency.

The problem to be solved is to provide histone deacetylase inhibitorswith high enzymatic and cellular activity that have increasedbioavailability and/or in vivo potency.

The novel compounds of the present invention solve the above-describedproblem. The compounds of the present invention show excellent histonedeacetylase inhibiting enzymatic and cellular activity. They have a highcapacity to activate the p21 gene. They can have a desirablepharmacokinetic profile and low affinity for the P450 enzymes, whichreduces the risk of adverse drug-drug interaction allowing also for awider safety margin.

Advantageous features of the present compounds can be metabolicstability, solubility and/or p21 induction capacity.

This invention concerns compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

-   n is 0 or 1 and when n is 0 than a direct bond is intended;-   m is 0, 1 or 2 and when n is 0 than a direct bond is intended;-   p is 0 or 1 and when n is 0 than a direct bond is intended;-   each X is independently N or CH;-   each Y is independently O, NH, N—C₁₋₆alkyl, CH or CH₂ and when Y is    CH then the substituent is attached to the Y atom of the ring    structure;-   R¹ is hydroxy or a radical of formula (a-1)

wherein

-   -   R⁹ is hydroxy or —NH₂;    -   R¹⁰ is hydrogen, thienyl, furanyl or phenyl and each thienyl,        furanyl or phenyl can optionally be substituted with halo,        amino, nitro, cyano, hydroxy, phenyl, C₁₋₆alkyl,        (diC₁₋₆alkyl)amino, C₁₋₆alkyloxy, phenylC₁₋₆alkyloxy,        hydroxyC₁₋₆alkyl, C₁₋₆alkyloxycarbonyl, hydroxycarbonyl,        C₁₋₆alkylcarbonyl, polyhaloC₁₋₆alkyloxy, polyhaloC₁₋₆alkyl,        C₁₋₆alkylsulfonyl, hydroxycarbonylC₁₋₆alkyl,        C₁₋₆alkylcarbonylamino, aminosulfonyl, aminosulfonylC₁₋₆alkyl,        isoxazolyl, aminocarbonyl, phenylC₂₋₆alkenyl, phenylC₃₋₆alkynyl        or pyridinylC₃₋₆alkynyl;    -   R⁶, R⁷ and R⁸ are each independently hydrogen, —NH₂, nitro,        furanyl, halo, C₁₋₆alkyl, C₁₋₆alkyloxy, trifluoromethyl,        thienyl, phenyl, C₁₋₆alkylcarbonylamino, aminocarbonylC₁₋₆alkyl        or —C≡C—CH₂—R¹¹;    -   wherein R¹¹ is hydrogen, C₁₋₆alkyl, hydroxy, amino or        C₁₋₆alkyloxy;

-   R² is C₁₋₆alkyl, C₃₋₇cycloalkyl, C₁₋₆alkylaminocarbonyl or    C₁₋₆alkyloxycarbonyl;

-   R³ is hydrogen, C₁₋₆alkyl, C₃₋₇cycloalkyl, hydroxyC₁₋₆alkyl,    C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxycarbonyl or    C₁₋₆alkylaminocarbonyl; or

-   R² and R³ can be bridged (i.e. forming a cyclic ring system) with a    methylene, ethylene or propylene bridge;

-   R⁴ is hydrogen, C₁₋₆alkyl, —C(═O)—CHR¹²R¹³ or —S(═O)₂—N(CH₃)₂;    wherein each R¹² and R¹³ is independently hydrogen, amino, C₁₋₆alkyl    or aminoC₁₋₆alkyl; and

-   R⁵ is hydrogen, hydroxy, amino, halo, C₁₋₆alkyl, polyhaloC₁₋₆alkyl,    C₁₋₆alkyloxycarbonyl, hydroxycarbonyl, C₁₋₆alkylcarbonylamino,    C₁₋₆alkyloxy, or mono- or di(C₁₋₆alkyl)amino.

Lines drawn into the bicyclic ring systems from substituents indicatethat the bonds may be attached to any of the suitable ring atoms of thebicyclic ring system.

The term “histone deacetylase inhibitor” or “inhibitor of histonedeacetylase” is used to identify a compound, which is capable ofinteracting with a histone deacetylase and inhibiting its activity, moreparticularly its enzymatic activity. Inhibiting histone deacetylaseenzymatic activity means reducing the ability of a histone deacetylaseto remove an acetyl group from a histone. Preferably, such inhibition isspecific, i.e. the histone deacetylase inhibitor reduces the ability ofa histone deacetylase to remove an acetyl group from a histone at aconcentration that is lower than the concentration of the inhibitor thatis required to produce some other, unrelated biological effect.

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₆alkyl defines straight and branchedchain saturated hydrocarbon radicals having from 1 to 6 carbon atomssuch as, e.g. methyl, ethyl, propyl, butyl, 1-methylethyl,2-methylpropyl, pentyl, 2-methyl-butyl, hexyl, 2-methylpentyl and thelike; C₂₋₆alkenyl defines straight and branched chain hydrocarbonradicals containing one double bond and having from 2 to 6 carbon atomssuch as, for example, ethenyl, 2-propenyl, 3-butenyl, 2-pentenyl,3-pentenyl, 3-methyl-2-butenyl, and the like; C₃₋₆alkynyl definesstraight and branch chained hydrocarbon radicals containing one triplebond and having from 3 to 6 carbon atoms, such as, for example,2-propynyl, 3-butynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 3-hexynyl, andthe like; polyhaloC₁₋₆alkyl defines C₁₋₆alkyl containing three identicalor different halo substituents for example trifluoromethyl; andC₃₋₇cycloalkyl includes cyclic hydrocarbon groups having from 3 to 6carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cycloheptyl and the like.

Pharmaceutically acceptable addition salts encompass pharmaceuticallyacceptable acid addition salts and pharmaceutically acceptable baseaddition salts. The pharmaceutically acceptable acid addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid addition salt forms, which the compounds of formula (I)are able to form. The compounds of formula (I) which have basicproperties can be converted in their pharmaceutically acceptable acidaddition salts by treating said base form with an appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric;nitric; phosphoric and the like acids; or organic acids such as, forexample, acetic, trifluoroacetic, propanoic, hydroxyacetic, lactic,pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-amino-salicylic, pamoic and the like acids. The compounds of formula(I) which have acidic properties may be converted in theirpharmaceutically acceptable base addition salts by treating said acidform with a suitable organic or inorganic base. Appropriate base saltforms comprise, for example, the ammonium salts, the alkali and earthalkaline metal salts, e.g. the lithium, sodium, potassium, magnesium,calcium salts and the like, salts with organic bases, e.g. thebenzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like. Theterm “acid or base addition salts” also comprises the hydrates and thesolvent addition forms, which the compounds of formula (I) are able toform. Examples of such forms are e.g. hydrates, alcoholates and thelike. The term “stereochemically isomeric forms of compounds of formula(I)”, as used herein, defines all possible compounds made up of the sameatoms bonded by the same sequence of bonds but having differentthree-dimensional structures, which are not interchangeable, which thecompounds of formula (I) may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms, which saidcompound may possess. Said mixture may contain all diastereomers and/orenantiomers of the basic molecular structure of said compound. Allstereochemically isomeric forms of the compounds of formula (I) both inpure form or in admixture with each other are intended to be embracedwithin the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide, particularly those N-oxides whereinone or more of the piperidine-, piperazine or pyridazinyl-nitrogens areN-oxidized. Some of the compounds of formula (I) may also exist in theirtautomeric forms. Such forms although not explicitly indicated in theabove formula are intended to be included within the scope of thepresent invention. Whenever used hereinafter, the term “compounds offormula (I)” is meant to include also the pharmaceutically acceptableaddition salts and all stereoisomeric forms. As used herein, the terms“histone deacetylase” and “HDAC” are intended to refer to any one of afamily of enzymes that remove acetyl groups from the ε-amino groups oflysine residues at the N-terminus of a histone. Unless otherwiseindicated by context, the term “histone” is meant to refer to anyhistone protein, including H1, H2A, H2B, H3, H4, and H5, from anyspecies. Human HDAC proteins or gene products, include, but are notlimited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7,HDAC-8, HDAC-9, HDAC-10 and HDAC-11. The histone deacetylase can also bederived from a protozoal or fungal source.

A first group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   -   a) n is 1;    -   b) p is 0;    -   c) each X is N;    -   d) each Y is CH;    -   e) R¹ is hydroxy;    -   f) R² is C₁₋₆alkyl;    -   g) R² and R³ can be bridged with a methylene, ethylene or        propylene bridge;    -   h) R³ is hydrogen;    -   i) R⁴ is C₁₋₆alkyl; and    -   j) R⁵ is hydrogen.

A second group of interesting compounds consists of those compounds offormula (I) in wherein one or more of the following restrictions apply:

-   -   a) n is 1;    -   b) p is 0;    -   c) each Y is CH;    -   d) R¹⁰ is hydrogen;    -   e) R⁶, R⁷ and R⁸ are each independently hydrogen,    -   f) R² is C₁₋₆alkyl;    -   g) R² and R³ can be bridged with a methylene, ethylene or        propylene bridge;    -   h) R³ is hydrogen;    -   i) R⁴ is C₁₋₆alkyl; and    -   j) R⁵ is hydrogen.

A third group of interesting compounds consists of those compounds offormula (I) or of those compounds of the first and second group whereinone or more of the following restriction apply:

-   -   a) n is 0;    -   b) each Y is independently O, CH or CH₂;    -   c) R¹ is hydroxy; and    -   d) R³ is C₃₋₇cycloalkyl, C₁₋₆alkyloxycarbonyl or        C₁₋₆alkylaminocarbonyl.

A fourth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   -   a) n is 1;    -   b) m is 1 or 2;    -   c) p is 0;    -   d) each X is N;    -   e) each Y is CH;    -   f) R² and R³ are bridged with a methylene, ethylene or propylene        bridge;    -   g) R⁴ is C₁₋₆alkyl;    -   h) R⁵ is hydrogen.

A group of preferred compounds consists of those compounds of formula(I) wherein n is 1; p is 0; each X is N; each Y is CH; R¹ is hydroxy; R²is C₁₋₆alkyl and R³ is hydrogen or R² and R³ can be bridged with amethylene, ethylene or propylene bridge; R⁴ is C₁₋₆alkyl; and R⁵ ishydrogen.

Another group of preferred compounds consists of those compounds offormula (I) wherein n is 1; p is 0; each Y is CH; R¹⁰ is hydrogen; R⁶,R⁷ and R⁸ are each independently hydrogen, R² is C₁₋₆alkyl and R³ ishydrogen or R² and R³ can be bridged with a methylene, ethylene orpropylene bridge; R⁴ is C₁₋₆alkyl; and R⁵ is hydrogen.

A group of more preferred compounds consists of those compounds offormula (I) wherein n is 1; m is 1 or 2; p is 0; each X is N; each Y isCH; R² and R³ are bridged with a methylene, ethylene or propylenebridge; R⁴ is C₁₋₆alkyl; and R⁵ is hydrogen. Most preferred compoundsare compound No. 2 and compound No. 3.

The compounds of formula (I) and their pharmaceutically acceptable saltsand N-oxides and stereochemically isomeric forms thereof may be preparedin conventional manner. The starting materials and some of theintermediates are known compounds and are commercially available or maybe prepared according to conventional reaction procedures generallyknown in the art or as described in patent applications EP1485099,EP1485348, EP1485353, EP1485354, EP1485364, EP1485365, EP1485370, andEP1485378.

Some preparation methods will be described hereinafter in more detail.Other methods for obtaining final compounds of formula (I) are describedin the examples.

Hydroxamic acids of formula (I), wherein R¹ is hydroxy, herein referredto as compounds of formula (I-a) may be prepared by reacting anintermediate of formula (II) with an appropriate acid, such as forexample, trifluoroacetic acid. Said reaction is performed in anappropriate solvent, such as, for example, methanol or dichloromethane.

Compounds of formula (I), wherein R¹ is a radical of formula (a-1) andR⁹ is —NH₂, herein referred to as compounds of formula (I-b) may beprepared by reacting an intermediate of formula (III) wherein Mrepresents hydrogen, or sodium or lithium or an alkali metal cation forexample sodium, herein referred to as compounds of formula (III-a), withan intermediate of formula (IV) in the presence of appropriate reagentssuch as for example benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP). The reaction may be performed in thepresence of a base such as triethylamine, in a suitable solvent, suchas, a mixture of dichloromethane and tetrahydrofuran.

Compounds of formula (I-b) may also be prepared by reacting anintermediate of formula (V) with an appropriate acid, such as forexample, trifluoroacetic acid. Said reaction is performed in anappropriate solvent, such as, for example, methanol or dichloromethane.

Compounds of formula (I), wherein R¹ is a radical of formula (a-1) andR⁹ is hydroxy, herein referred to as compounds of formula (I-c) may beprepared by reacting an intermediate of formula (VI) withtetrabutylammonium fluoride in an appropriate solvent such as, forexample tetrahydrofuran. TBDMS in the intermediate of formula (VI) meanstert-butyl(dimethyl)silanyl.

Intermediates of formula (II) may be prepared by reacting anintermediate of formula (III) with an intermediate of formula (VII) inthe presence of appropriate reagents such asN′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). Thereaction may be performed in the presence of a base such astriethylamine, in a suitable solvent, such as, a mixture ofdichloromethane and tetrahydrofuran.

Intermediates of formula (V) may be prepared by reacting an intermediateof formula (III) with an appropriate tert-butyloxycarbonyl (Boc)protected phenylamine of formula (VIII) in the presence ofbenzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate(BOP) and sodium hydride. Said reaction can be performed in a suitablesolvent such as, for example, pyridine.

Intermediates of formula (VI) may be prepared by reacting anintermediate of formula (III) with an appropriatetert-butyl(dimethyl)silanyl (TBDMS) protected phenylamine of formula(IX) in the presence ofbenzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate(BOP) and triethylamine. Said reaction is performed in a suitablesolvent such as, for example, N,N-dimethylformamide.

Intermediates of formula (III) may be prepared by reacting anintermediate of formula (X) with an appropriate acidic solution, e.g.hydrochloric acid, or basic solution, e.g. lithiumhydroxide orsodiumhydroxide, in a suitable solvent e.g. an alcohol, such as ethanolor propanol or dioxane.

Intermediates of formula (X) wherein R³ is as defined above other thanhydrogen may be prepared by reacting a corresponding compound of formula(X) wherein R³ is hydrogen, with an appropriate functional reagentserving to introduce the appropriate R³ group. For example when R³ isC₁₋₆alkyl, C₃₋₇cycloalkyl, hydroxyC₁₋₆alkyl or C₁₋₆alkyloxyC₁₋₆alkylthen the reagent is an appropriate alkylating agent for example anappropriate alkyl halide, e.g. chloride, the reaction being effectedunder basic conditions for example using triethylamine, in a suitablesolvent such as dimethylformamide. When R³ is C₁₋₆alkyloxycarbonyl orC₁₋₆alkylaminocarbonyl then the reagent is an appropriate acylatingagent for example an appropriate acyl halide, e.g. chloride, thereaction being effected under basic conditions for example usingtriethylamine, in a suitable solvent such as dichloromethane.

Intermediates of formula (X) wherein R³ is hydrogen, herein referred toas intermediates of formula (X-a) may be prepared by reacting anintermediate of formula (XI) with an intermediate of formula (XII) inthe presence of appropriate reagents, such as NaBH(OAc)₃ and Ti(OEt)₄,in a suitable solvent such as dichloroethane or methanol. Alternatively,the reaction can be effected using BH₃CN under acidic conditions e.g.using acetic acid in a suitable solvent such as methanol, or underhydrogenating conditions using a palladium-carbon catalyst in a suitablesolvent such as methanol.

Intermediates of formula (X) wherein n is 1, R² and R³ are bridged, thedotted line represents a methylene, ethylene or propylene bridge and nis 1, herein referred to as intermediates of formula (X-b), may beprepared by reacting an intermediate of formula (XIII) with anintermediate of formula (XIV) in the presence of NaBH(OAc)₃ and Ti(OEt)₄in a suitable solvent such as dichloroethane.

Alternatively, the reaction can be effected using BH₃CN under acidicconditions e.g. using acetic acid in a suitable solvent such asmethanol, or under hydrogenating conditions using a palladium-carboncatalyst in a suitable solvent such as methanol.

The present invention also concerns intermediates of formula (II),(III), (X), (X-a) and (X-b) referred to collectively as compounds offormula (A) in which Q is C₁₋₂alkyloxycarbonyl, hydroxycarbonyl ortetrahydropyranyloxyaminocarbonyl and n, m, p, X, Y, Z, R², R³, R⁴ andR⁵ are as defined for formula (I), and the N-oxide forms, thepharmaceutically acceptable addition salts and the stereo-chemicallyisomeric forms thereof.

Groups of interesting, preferred, more preferred and most preferredcompounds can be defined for the above intermediates, in accordance withthe groups defined for the compounds of formula (I).

The compounds of formula (I) and some of the intermediates may have atleast one stereogenic centre in their structure. This stereogenic centremay be present in an R or an S configuration.

The compounds of formula (I) as prepared in the hereinabove describedprocesses are generally racemic mixtures of enantiomers, which can beseparated from one another following art-known resolution procedures.The racemic compounds of formula (I) may be converted into thecorresponding diastereomeric salt forms by reaction with a suitablechiral acid. Said diastereomeric salt forms are subsequently separated,for example, by selective or fractional crystallization and theenantiomers are liberated there from by alkali. An alternative manner ofseparating the enantiomeric forms of the compounds of formula (I)involves liquid chromatography using a chiral stationary phase. Saidpure stereochemically isomeric forms may also be derived from thecorresponding pure stereochemically isomeric forms of the appropriatestarting materials, provided that the reaction occursstereospecifically. Preferably if a specific stereoisomer is desired,said compound would be synthesized by stereospecific methods ofpreparation. These methods will advantageously employ enantiomericallypure starting materials.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof have valuablepharmacological properties in that they have a histone deacetylase(HDAC) inhibitory effect.

This invention provides a method for inhibiting the abnormal growth ofcells, including transformed cells, by administering an effective amountof a compound of the invention. Abnormal growth of cells refers to cellgrowth independent of normal regulatory mechanisms (e.g. loss of contactinhibition). This includes the inhibition of tumour growth both directlyby causing growth arrest, terminal differentiation and/or apoptosis ofcancer cells, and indirectly, by inhibiting neovascularization oftumours.

This invention also provides a method for inhibiting tumour growth byadministering an effective amount of a compound of the presentinvention, to a subject, e.g. a mammal (and more particularly a human)in need of such treatment. In particular, this invention provides amethod for inhibiting the growth of tumours by the administration of aneffective amount of the compounds of the present invention. Examples oftumours which may be inhibited, but are not limited to, lung cancer(e.g. adenocarcinoma and including non-small cell lung cancer),pancreatic cancers (e.g. pancreatic carcinoma such as, for exampleexocrine pancreatic carcinoma), colon cancers (e.g. colorectalcarcinomas, such as, for example, colon adenocarcinoma and colonadenoma), prostate cancer including the advanced disease, hematopoietictumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-celllymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acutemyelogenous leukemia (AML)), thyroid follicular cancer, myelodysplasticsyndrome (MDS), tumours of mesenchymal origin (e.g. fibrosarcomas andrhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas,gliomas, benign tumour of the skin (e.g. keratoacanthomas), breastcarcinoma (e.g. advanced breast cancer), kidney carcinoma, ovarycarcinoma, bladder carcinoma and epidermal carcinoma.

The compound according to the invention may be used for othertherapeutic purposes, for example:

-   -   a) the sensitisation of tumours to radiotherapy by administering        the compound according to the invention before, during or after        irradiation of the tumour for treating cancer;    -   b) treating arthropathies and osteopathological conditions such        as rheumatoid arthritis, osteoarthritis, juvenile arthritis,        gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis        and systemic lupus erythematosus;    -   c) inhibiting smooth muscle cell proliferation including        vascular proliferative disorders, atherosclerosis and        restenosis;    -   d) treating inflammatory conditions and dermal conditions such        as ulcerative colitis, Crohn's disease, allergic rhinitis, graft        vs. host disease, conjunctivitis, asthma, ARDS, Behcets disease,        transplant rejection, uticaria, allergic dermatitis, alopecia        greata, scleroderma, exanthema, eczema, dermatomyositis, acne,        diabetes, systemic lupus erythematosis, Kawasaki's disease,        multiple sclerosis, emphysema, cystic fibrosis and chronic        bronchitis;    -   e) treating endometriosis, uterine fibroids, dysfunctional        uterine bleeding and endometrial hyperplasia;    -   f) treating ocular vascularisation including vasculopathy        affecting retinal and choroidal vessels;    -   g) treating a cardiac dysfunction;    -   h) inhibiting immunosuppressive conditions such as the treatment        of HIV infections;    -   i) treating renal dysfunction;    -   j) suppressing endocrine disorders;    -   k) inhibiting dysfunction of gluconeogenesis;    -   l) treating a neuropathology for example Parkinson's disease or        a neuropathology that results in a cognitive disorder, for        example, Alzheimer's disease or polyglutamine related neuronal        diseases;    -   m) treating psychiatric disorders for example schizophrenia,        bipolar disorder, depression, anxiety and psychosis;    -   n) inhibiting a neuromuscular pathology, for example,        amylotrophic lateral sclerosis;    -   o) treating spinal muscular atrophy;    -   p) treating other pathologic conditions amenable to treatment by        potentiating expression of a gene;    -   q) enhancing gene therapy;    -   r) inhibiting adipogenesis;    -   s) treating parasitosis such as malaria.

Hence, the present invention discloses the compounds of formula (I) foruse as a medicine as well as the use of these compounds of formula (I)for the manufacture of a medicament for treating one or more of theabove mentioned conditions.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof can have valuablediagnostic properties in that they can be used for detecting oridentifying a HDAC in a biological sample comprising detecting ormeasuring the formation of a complex between a labelled compound and aHDAC.

The detecting or identifying methods can use compounds that are labelledwith labelling agents such as radioisotopes, enzymes, fluorescentsubstances, luminous substances, etc. Examples of the radioisotopesinclude ¹²⁵I, ¹³¹I, ³H and ¹⁴C. Enzymes are usually made detectable byconjugation of an appropriate substrate which, in turn catalyses adetectable reaction. Examples thereof include, for example,beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidaseand malate dehydrogenase, preferably horseradish peroxidase. Theluminous substances include, for example, luminol, luminol derivatives,luciferin, aequorin and luciferase.

Biological samples can be defined as body tissue or body fluids.Examples of body fluids are cerebrospinal fluid, blood, plasma, serum,urine, sputum, saliva and the like.

In view of their useful pharmacological properties, the subjectcompounds may be formulated into various pharmaceutical forms foradministration purposes.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. For parenteralcompositions, the carrier will usually comprise sterile water, at leastin large part, though other ingredients, to aid solubility for example,may be included. Injectable solutions, for example, may be prepared inwhich the carrier comprises saline solution, glucose solution or amixture of saline and glucose solution. Injectable suspensions may alsobe prepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. In the compositions suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not cause a significant deleterious effect to theskin. Said additives may facilitate the administration to the skinand/or may be helpful for preparing the desired compositions. Thesecompositions may be administered in various ways, e.g., as a transdermalpatch, as a spot-on or as an ointment.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used in thespecification and claims herein refers to physically discrete unitssuitable as unitary dosages, each unit containing a predeterminedquantity of active ingredient, calculated to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier. Examples of such dosage unit forms are tablets (includingscored or coated tablets), capsules, pills, powder packets, wafers,injectable solutions or suspensions, teaspoonfuls, tablespoonfuls andthe like, and segregated multiples thereof.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that a therapeutically effective amount would be from 0.005mg/kg to 100 mg/kg body weight, and in particular from 0.005 mg/kg to 10mg/kg body weight. It may be appropriate to administer the required doseas two, three, four or more sub-doses at appropriate intervalsthroughout the day. Said sub-doses may be formulated as unit dosageforms, for example, containing 0.5 to 500 mg, and in particular 10 mg to500 mg of active ingredient per unit dosage form.

As another aspect of the present invention a combination of aHDAC-inhibitor with another anticancer agent is envisaged, especiallyfor use as a medicine, more specifically in the treatment of cancer orrelated diseases.

For the treatment of the above conditions, the compounds of theinvention may be advantageously employed in combination with one or moreother medicinal agents, more particularly, with other anti-canceragents. Examples of anti-cancer agents are:

-   -   platinum coordination compounds for example cisplatin,        carboplatin or oxaliplatin;    -   taxane compounds for example paclitaxel or docetaxel;    -   topoisomerase I inhibitors such as camptothecin compounds for        example irinotecan or topotecan;    -   topoisomerase II inhibitors such as anti-tumour podophyllotoxin        derivatives for example etoposide or teniposide;    -   anti-tumour vinca alkaloids for example vinblastine, vincristine        or vinorelbine;    -   anti-tumour nucleoside derivatives for example 5-fluorouracil,        gemcitabine or capecitabine;    -   alkylating agents such as nitrogen mustard or nitrosourea for        example cyclophosphamide, chlorambucil, carmustine or lomustine;    -   anti-tumour anthracycline derivatives for example daunorubicin,        doxorubicin, idarubicin or mitoxantrone;    -   HER2 antibodies for example trastuzumab;    -   estrogen receptor antagonists or selective estrogen receptor        modulators for example tamoxifen, toremifene, droloxifene,        faslodex or raloxifene;    -   aromatase inhibitors such as exemestane, anastrozole, letrazole        and vorozole;    -   differentiating agents such as retinoids, vitamin D and retinoic        acid metabolism blocking agents (RAMBA) for example accutane;    -   DNA methyl transferase inhibitors for example azacytidine;    -   kinase inhibitors for example flavopiridol, imatinib mesylate or        gefitinib;    -   farnesyltransferase inhibitors;    -   other HDAC inhibitors    -   inhibitors of the ubiquitin-proteasome pathway for example        Velcade; or    -   Yondelis.

The term “platinum coordination compound” is used herein to denote anytumour cell growth inhibiting platinum coordination compound whichprovides platinum in the form of an ion.

The term “taxane compounds” indicates a class of compounds having thetaxane ring system and related to or derived from extracts from certainspecies of yew (Taxus) trees.

The term “topoisomerase inhibitors” is used to indicate enzymes that arecapable of altering DNA topology in eukaryotic cells. They are criticalfor important cellular functions and cell proliferation. There are twoclasses of topoisomerases in eukaryotic cells, namely type I and typeII. Topoisomerase I is a monomeric enzyme of approximately 100,000molecular weight. The enzyme binds to DNA and introduces a transientsingle-strand break, unwinds the double helix (or allows it to unwind)and subsequently reseals the break before dissociating from the DNAstrand. Topisomerase II has a similar mechanism of action which involvesthe induction of DNA strand breaks or the formation of free radicals.

The term “camptothecin compounds” is used to indicate compounds that arerelated to or derived from the parent camptothecin compound which is awater-insoluble alkaloid derived from the Chinese tree Camptothecinacuminata and the Indian tree Nothapodytes foetida.

The term “podophyllotoxin compounds” is used to indicate compounds thatare related to or derived from the parent podophyllotoxin, which isextracted from the mandrake plant.

The term “anti-tumour vinca alkaloids” is used to indicate compoundsthat are related to or derived from extracts of the periwinkle plant(Vinca rosea).

The term “alkylating agents” encompass a diverse group of chemicals thathave the common feature that they have the capacity to contribute, underphysiological conditions, alkyl groups to biologically vitalmacromolecules such as DNA. With most of the more important agents suchas the nitrogen mustards and the nitrosoureas, the active alkylatingmoieties are generated in vivo after complex degradative reactions, someof which are enzymatic. The most important pharmacological actions ofthe alkylating agents are those that disturb the fundamental mechanismsconcerned with cell proliferation in particular DNA synthesis and celldivision. The capacity of alkylating agents to interfere with DNAfunction and integrity in rapidly proliferating tissues provides thebasis for their therapeutic applications and for many of their toxicproperties.

The term “anti-tumour anthracycline derivatives” comprise antibioticsobtained from the fungus Strep. peuticus var. caesius and theirderivatives, characterised by having a tetracycline ring structure withan unusual sugar, daunosamine, attached by a glycosidic linkage.

Amplification of the human epidermal growth factor receptor 2 protein(HER 2) in primary breast carcinomas has been shown to correlate with apoor clinical prognosis for certain patients. Trastuzumab is a highlypurified recombinant DNA-derived humanized monoclonal IgG1 kappaantibody that binds with high affinity and specificity to theextracellular domain of the HER2 receptor.

Many breast cancers have estrogen receptors and growth of these tumourscan be stimulated by estrogen. The terms “estrogen receptor antagonists”and “selective estrogen receptor modulators” are used to indicatecompetitive inhibitors of estradiol binding to the estrogen receptor(ER). Selective estrogen receptor modulators, when bound to the ER,induces a change in the three-dimensional shape of the receptor,modulating its binding to the estrogen responsive element (ERE) on DNA.

In postmenopausal women, the principal source of circulating estrogen isfrom conversion of adrenal and ovarian androgens (adrostenedione andtestosterone) to estrogens (estrone and estradiol) by the aromataseenzyme in peripheral tissues. Estrogen deprivation through aromataseinhibition or inactivation is an effective and selective treatment forsome postmenopausal patients with hormone-dependent breast cancer.

The term “antiestrogen agent” is used herein to include not onlyestrogen receptor antagonists and selective estrogen receptor modulatorsbut also aromatase inhibitors as discussed above.

The term “differentiating agents” encompass compounds that can, invarious ways, inhibit cell proliferation and induce differentiation.Vitamin D and retinoids are known to play a major role in regulatinggrowth and differentiation of a wide variety of normal and malignantcell types. Retinoic acid metabolism blocking agents (RAMBA's) increasethe levels of endogenous retinoic acids by inhibiting the cytochromeP450-mediated catabolism of retinoic acids.

DNA methylation changes are among the most common abnormalities in humanneoplasia. Hypermethylation within the promoters of selected genes isusually associated with inactivation of the involved genes. The term“DNA methyl transferase inhibitors” is used to indicate compounds thatact through pharmacological inhibition of DNA methyl transferase andreactivation of tumour suppressor gene expression.

The term “kinase inhibitors” comprises potent inhibitors of kinases thatare involved in cell cycle progression and programmed cell death(apoptosis)

The term “farnesyltransferase inhibitors” is used to indicate compoundsthat were designed to prevent farnesylation of Ras and otherintracellular proteins. They have been shown to have effect on malignantcell proliferation and survival.

The term “other HDAC inhibitors” comprises but is not limited to:

-   -   carboxylates for example butyrate, cinnamic acid,        4-phenylbutyrate or valproic acid;    -   hydroxamic acids for example suberoylanilide hydroxamic acid        (SAHA), piperazine containing SAHA analogues, biaryl hydroxamate        A-161906 and its carbozolylether-, tetrahydropyridine- and        tetralone-analogues, bicyclic aryl-N-hydroxycarboxamides,        pyroxamide, CG-1521, PXD-101, sulfonamide hydroxamic acid,        LAQ-824, LBH-589, trichostatin A (TSA), oxamflatin, scriptaid,        scriptaid related tricyclic molecules, m-carboxy cinnamic acid        bishydroxamic acid (CBHA), CBHA-like hydroxamic acids,        trapoxin-hydroxamic acid analogue, CRA-024781, R306465 and        related benzoyl- and heteroaryl-hydroxamic acids, aminosuberates        and malonyldiamides;    -   cyclic tetrapeptides for example trapoxin, apidicin,        depsipeptide, spiruchostatin-related compounds, RedFK-228,        sulfhydryl-containing cyclic tetrapeptides (SCOPs), hydroxamic        acid containing cyclic tetrapeptides (CHAPs), TAN-174s and        azumamides;    -   benzamides for example MS-275 or CI-994, or    -   depudecin.

The term “inhibitors of the ubiquitin-proteasome pathway” is used toidentify compounds that inhibit the targeted destruction of cellularproteins in the proteasome, including cell cycle regulatory proteins.

For the treatment of cancer the compounds according to the presentinvention may be administered to a patient as described above, inconjunction with irradiation. Irradiation means ionising radiation andin particular gamma radiation, especially that emitted by linearaccelerators or by radionuclides that are in common use today. Theirradiation of the tumour by radionuclides can be external or internal.

The present invention also relates to a combination according to theinvention of an anti-cancer agent and a HDAC inhibitor according to theinvention.

The present invention also relates to a combination according to theinvention for use in medical therapy for example for inhibiting thegrowth of tumour cells.

The present invention also relates to a combinations according to theinvention for inhibiting the growth of tumour cells.

The present invention also relates to a method of inhibiting the growthof tumour cells in a human subject which comprises administering to thesubject an effective amount of a combination according to the invention.

This invention further provides a method for inhibiting the abnormalgrowth of cells, including transformed cells, by administering aneffective amount of a combination according to the invention.

The other medicinal agent and HDAC inhibitor may be administeredsimultaneously (e.g. in separate or unitary compositions) orsequentially in either order. In the latter case, the two compounds willbe administered within a period and in an amount and manner that issufficient to ensure that an advantageous or synergistic effect isachieved. It will be appreciated that the preferred method and order ofadministration and the respective dosage amounts and regimes for eachcomponent of the combination will depend on the particular othermedicinal agent and HDAC inhibitor being administered, their route ofadministration, the particular tumour being treated and the particularhost being treated. The optimum method and order of administration andthe dosage amounts and regime can be readily determined by those skilledin the art using conventional methods and in view of the information setout herein.

The platinum coordination compound is advantageously administered in adosage of 1 to 500 mg per square meter (mg/m²) of body surface area, forexample 50 to 400 mg/m², particularly for cisplatin in a dosage of about75 mg/m² and for carboplatin in about 300 mg/m² per course of treatment.

The taxane compound is advantageously administered in a dosage of 50 to400 mg per square meter (mg/m²) of body surface area, for example 75 to250 mg/m², particularly for paclitaxel in a dosage of about 175 to 250mg/m² and for docetaxel in about 75 to 150 mg/m² per course oftreatment.

The camptothecin compound is advantageously administered in a dosage of0.1 to 400 mg per square meter (mg/m²) of body surface area, for example1 to 300 mg/m², particularly for irinotecan in a dosage of about 100 to350 mg/m² and for topotecan in about 1 to 2 mg/m² per course oftreatment.

The anti-tumour podophyllotoxin derivative is advantageouslyadministered in a dosage of 30 to 300 mg per square meter (mg/m²) ofbody surface area, for example 50 to 250 mg/m², particularly foretoposide in a dosage of about 35 to 100 mg/m² and for teniposide inabout 50 to 250 mg/m² per course of treatment.

The anti-tumour vinca alkaloid is advantageously administered in adosage of 2 to 30 mg per square meter (mg/m²) of body surface area,particularly for vinblastine in a dosage of about 3 to 12 mg/m², forvincristine in a dosage of about 1 to 2 mg/m², and for vinorelbine indosage of about 10 to 30 mg/m² per course of treatment.

The anti-tumour nucleoside derivative is advantageously administered ina dosage of 200 to 2500 mg per square meter (mg/m²) of body surfacearea, for example 700 to 1500 mg/m², particularly for 5-FU in a dosageof 200 to 500 mg/m², for gemcitabine in a dosage of about 800 to 1200mg/m² and for capecitabine in about 1000 to 2500 mg/m² per course oftreatment.

The alkylating agents such as nitrogen mustard or nitrosourea isadvantageously administered in a dosage of 100 to 500 mg per squaremeter (mg/m²) of body surface area, for example 120 to 200 mg/m²,particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m²,for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg, for carmustinein a dosage of about 150 to 200 mg/m², and for lomustine in a dosage ofabout 100 to 150 mg/m² per course of treatment.

The anti-tumour anthracycline derivative is advantageously administeredin a dosage of 10 to 75 mg per square meter (mg/m²) of body surfacearea, for example 15 to 60 mg/m², particularly for doxorubicin in adosage of about 40 to 75 mg/m², for daunorubicin in a dosage of about 25to 45 mg/m², and for idarubicin in a dosage of about 10 to 15 mg/m² percourse of treatment.

Trastuzumab is advantageously administered in a dosage of 1 to 5 mg persquare meter (mg/m²) of body surface area, particularly 2 to 4 mg/m² percourse of treatment.

The antiestrogen agent is advantageously administered in a dosage ofabout 1 to 100 mg daily depending on the particular agent and thecondition being treated. Tamoxifen is advantageously administered orallyin a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day,continuing the therapy for sufficient time to achieve and maintain atherapeutic effect. Toremifene is advantageously administered orally ina dosage of about 60 mg once a day, continuing the therapy forsufficient time to achieve and maintain a therapeutic effect.Anastrozole is advantageously administered orally in a dosage of about 1mg once a day. Droloxifene is advantageously administered orally in adosage of about 20-100 mg once a day. Raloxifene is advantageouslyadministered orally in a dosage of about 60 mg once a day. Exemestane isadvantageously administered orally in a dosage of about 25 mg once aday.

These dosages may be administered for example once, twice or more percourse of treatment, which may be repeated for example every 7, 14, 21or 28 days.

In view of their useful pharmacological properties, the components ofthe combinations according to the invention, i.e. the other medicinalagent and the HDAC inhibitor may be formulated into variouspharmaceutical forms for administration purposes. The components may beformulated separately in individual pharmaceutical compositions or in aunitary pharmaceutical composition containing both components.

The present invention therefore also relates to a pharmaceuticalcomposition comprising the other medicinal agent and the HDAC inhibitortogether with one or more pharmaceutical carriers.

The present invention also relates to a combination according to theinvention in the form of a pharmaceutical composition comprising ananti-cancer agent and a HDAC inhibitor according to the inventiontogether with one or more pharmaceutical carriers.

The present invention further relates to the use of a combinationaccording to the invention in the manufacture of a pharmaceuticalcomposition for inhibiting the growth of tumour cells.

The present invention further relates to a product containing as firstactive ingredient a HDAC inhibitor according to the invention and assecond active ingredient an anticancer agent, as a combined preparationfor simultaneous, separate or sequential use in the treatment ofpatients suffering from cancer.

EXPERIMENTAL PART

The following examples illustrate the present invention.

Hereinafter, “EDC” is defined asN′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride “DCM” is defined as dichloromethane, “DMSO” is definedas dimethyl sulfoxide, “EtOAc” is defined as ethyl acetate, “EtOH” isdefined as ethanol, “HOBt” is defined as 1-hydroxy-1H-benzotriazole,“MeOH” is defined as methanol, “TFA” is defined as trifluoroacetic acidand “THF” is defined as tetrahydrofuran.

A. Preparation of the Intermediate Compounds Example A1 a) Preparationof Intermediate 1

Titanium (IV) ethoxide (0.017 mol) was added at room temperature to asolution of 2-[4-(aminomethyl)-1-piperidinyl]-5-pyrimidinecarboxylicacid, ethyl ester (0.0083 mol) and 1-(1-methyl-1H-indol-3-yl)-ethanone(0.01 mol) in 1,2-dichloro-ethane (150 ml) under N₂ flow. The mixturewas stirred at room temperature for 4 days. Tris(acetato-α-O)hydroborate (1-), sodium (0.017 mol) was added. The mixture was stirredat room temperature for 3 days, poured out into potassium carbonate 10%.DCM was added. The mixture was stirred at room temperature for 1 hour,then filtered over celite. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (5 g) waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/MeOH/NH₄OH 97/3/1). The pure fractions were collected and thesolvent was evaporated, yielding 1.9 g (54%) of intermediate 1.

b) Preparation of Intermediate 2

A mixture of intermediate 1 (0.0045 mol) and sodium hydroxide (0.018mol) in EtOH (190 ml) was stirred and refluxed for 4 hours, then cooledto room temperature and evaporated till dryness. The residue was takenup in diethyl ether. The precipitate was filtered off and dried,yielding 1.6 g (86%) of intermediate 2, melting point >260° C.

c.) Preparation of Intermediate 3

EDC (0.0117 mol) and HOBT (0.0117 mol) were added to a solution ofintermediate 2 (0.0039 mol) in THF (160 ml) and DCM (160 ml) under N₂flow. The mixture was stirred at room temperature for 30 minutes.O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0117 mol) was added. Themixture was stirred at room temperature for 3 days, poured out into icewater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (3 g) waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/MeOH/NH₄OH 93/7/0.5). The pure fractions were collected and thesolvent was evaporated, yielding 1.55 g (82%) of intermediate 3.

Example A2 a.) Preparation of Intermediate 4

Sodium cyanoborohydride (0.0034 mol) and acetic acid (0.037 mol) wereadded at room temperature to a solution of intermediate 8 (0.0038 mol)and 1-methyl-3-(3-pyrrolidinyl)-1H-indole (0.0034 mol) in MeOH (70 ml)under N₂ flow. The mixture was stirred at room temperature for 48 hours,poured out into potassium carbonate 10% and extracted with DCM. Theorganic layer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue (1.6 g) was purified by column chromatographyover silica gel (5 μm) (eluent: DCM/MeOH/NH₄OH 99/1/0.05 to 94/6/0.3).The pure fractions were collected and the solvent was evaporated,yielding 0.38 g (26%) of intermediate 4.

b) Preparation of Intermediate 5

A mixture of intermediate 4 (0.0096 mol) and sodium hydroxide (0.038mol) in EtOH (450 ml) was stirred and refluxed for 4 hours, then cooledto room temperature and taken up in diethyl ether. The precipitate wasfiltered off and dried, yielding 3.8 g (90%) of intermediate 5, meltingpoint >260° C.

c) Preparation of Intermediate 6

EDC (0.0172 mol) and HOBT (0.0172 mol) were added at room temperature toa solution of intermediate 5 (0.0086 mol) in THF (400 ml) and DCM (400ml) under N₂ flow. The mixture was stirred for 45 minutes.O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0172 mol) was added. Themixture was stirred at room temperature for 24 hours, poured out intoice water and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (6.8 g)was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/MeOH/NH₄OH 95/5/0.1). The pure fractions were collected andthe solvent was evaporated, yielding 3 g (68%) of intermediate 6,melting point 80° C.

Example A3 a) Preparation of Intermediate 7

A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethylester (0.094 mol) in acetonitrile (40 ml) was added at 110° C. to asuspension of 4-piperidinemethanol (0.086 mol) and potassium carbonate(0.172 mol) in acetonitrile (200 ml) under N₂ flow. The mixture wasbrought to room temperature, then stirred for 4 hours, poured out intowater and extracted with EtOAc. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (23 g)was crystallized from CH₃CN/diethyl ether. The precipitate was filteredoff and dried, yielding 7.8 g (34%) of intermediate 7. The residue waspurified by column chromatography over silica gel (20-45 μm) (eluent:DCM/MeOH/NH₄OH 97/3/0.1). The pure fractions were collected and thesolvent was evaporated, yielding 4.6 g (20%) of intermediate 7, meltingpoint 129° C.

b) Preparation of Intermediate 8

DMSO (0.127 mol) was added at −78° C. to a solution of ethanedioldichloride (0.061 mol) in DCM (10 ml) under N₂ flow. The mixture wasstirred for 15 minutes. A solution of intermediate 7 (0.051 mol) in DCM(200 ml) was added. The mixture was stirred at −78° C. for 1 hour and 30minutes. Triethylamine (0.26 mol) was added dropwise. The mixture wasstirred at −78° C. for 15 minutes, then cooled to room temperature for 2hours and 30 minutes. Water was added. The mixture was extracted withDCM. The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (14 g) was purified by columnchromatography over silica gel (20-45 μm)(eluent: cyclohexane/EtOAc70/30). The pure fractions were collected and the solvent wasevaporated, yielding 7.6 g (57%) of intermediate 8.

c) Preparation of Intermediate 9

Titanium (IV) ethoxide (0.0019 mol) was added at room temperature to asolution of intermediate 8 (0.0014 mol) and1-methyl-3-(3-piperidinyl)-1H-indole (0.0012 mol) in 1,2-dichloro-ethane(25 ml) under N₂ flow. The mixture was stirred at 60° C. for 24 hours,then cooled to 5° C. Tris(acetato-α-O) hydroborate (1-), sodium (0.0036mol) was added under N₂ flow. The mixture was stirred at roomtemperature for 48 hours, poured out into potassium carbonate 10%. DCMwas added. The mixture was filtered over celite. The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue (1 g) was purified by column chromatography over silica gel(15-40 μm) (eluent: DCM/MeOH/NH₄OH 97/3/0.1). The pure fractions werecollected and the solvent was evaporated, yielding 0.36 g (44%) ofintermediate 9.

d) Preparation of Intermediate 10

A mixture of intermediate 9 (0.0008 mol) and sodium hydroxide (0.0032mol) in EtOH (40 ml) was stirred and refluxed for 4 hours and 30minutes, then cooled to room temperature and evaporated till dryness.The residue was taken up in diethyl ether. The precipitate was filteredoff and dried, yielding 0.36 g (100%) of intermediate 10, meltingpoint >260° C.

e) Preparation of Intermediate 11

EDC (0.0024 mol) and HOBT (0.0024 mol) were added at room temperature toa solution of intermediate 10 (0.0008 mol) in THF (40 ml) and DCM (40ml) under N₂ flow. The mixture was stirred for 15 minutes.O-(tetrahydro-2H-pyran-2-yl)—hydroxylamine (0.0024 mol) was added. Themixture was stirred at room temperature for 4 days, poured out intowater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated. The residue (0.49 g)was purified by column chromatography over kromasil (5 μm) (eluent:DCM/MeOH/NH₄OH 98/2/0.2 to 93/7/0.7). The pure fractions were collectedand the solvent was evaporated, yielding 0.26 g (61%) of intermediate11.

Example A4 a) Preparation of Intermediate 12

Ethanol, titanium(4+) salt (0.0172 mol) was added at room temperature toa solution of intermediate 8 (0.0104 mol) and1-methyl-3-(3-pyrrolidinyl)-1H-indole (0.0115 mol) in 1,2-dichloroethane(50 ml) under N₂ flow. The mixture was stirred at 60° C. for 3 hours,then stirred at room temperature overnight. Tris(acetato-α-O)hydroborate (1-), sodium (0.0345 mol) was added portionwise. The mixturewas stirred at room temperature overnight and poured out into ice water.DCM was added. The mixture was filtered over celite. The filtrate wasextracted with DCM. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue (3.2 g) waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/MeOH/NH₄OH 96/4/0.2). The pure fractions were collected and thesolvent was evaporated, yielding 0.55 g (11%) of intermediate 12.

b) Preparation of Intermediate 13

A mixture of intermediate 12 (0.0012 mol) and sodium hydroxide (0.0024mol) in EtOH (40 ml) was stirred at 60° C. for 8 hours, then cooled toroom temperature. The precipitate was filtered, washed with EtOH, thenwith diethyl ether and dried, yielding 0.6 g (100%) of intermediate 13,isolated as a sodium salt.

c) Preparation of Intermediate 14

HOBt (0.0024 mol) and EDC (0.0024 mol) were added at room temperature toa solution of intermediate 13 (0.0016 mol) and triethylamine (0.0048mol) in DCM/THF (20 ml). The mixture was stirred at room temperature for15 minutes. O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0032 mol) wasadded. The mixture was stirred at room temperature for 48 hours, pouredout into water and extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue (0.6g) was purified by column chromatography over kromasil (10 μm) (eluent:DCM/MeOH/NH4OH 97/3/0.3). The pure fractions were collected and thesolvent was evaporated, yielding 0.046 g (6%) of intermediate 14.

B. Preparation of the Final Compounds Example B1 Preparation ofIntermediate 1

A mixture of intermediate 3 (0.003 mol) in TFA (7.5 ml) and MeOH (150ml) was stirred at room temperature for 24 hours, then evaporated at 30°C. The residue was purified by column chromatography over amino coatedsilica gel (25-40 μm) (eluent: DCM/MeOH/water 80/20/2). The purefractions were collected and the solvent was evaporated. The residue(0.453 g, 37%) was crystallized from MeOH/CH₃CN/diethyl ether. Theprecipitate was filtered off and dried, yielding 0.255 g (18%) ofcompound 1, melting point 296° C.

Example B2 Preparation of Intermediate 2

A mixture of intermediate 6 (0.0003 mol) in TFA (0.9 ml) and MeOH (18ml) was stirred at room temperature for 24 hours, then evaporated tilldryness. The residue was purified by column chromatography over aminocoated silica gel (25-40 μm)(eluent: DCM/MeOH/water 90/10/1). The purefractions were collected and the solvent was evaporated. The residue(0.195 g) was taken up several times with diethyl ether. The mixture wasevaporated and dried, yielding 0.122 g (66%) of compound 2, meltingpoint 231° C.

Example B3 Preparation of Compound 3

A mixture of intermediate 11 (0.0005 mol) in TFA (1.5 ml) and MeOH (30ml) was stirred at room temperature for 24 hours, then evaporated at 30°C. The residue was crystallized from CH₃CN/MeOH/diethyl ether. Theprecipitate was filtered off and dried, yielding 0.17 g (56%) ofcompound 3, melting point 224° C.

Example B4 Preparation of Compound 4

A mixture of intermediate 14 (0.00009 mol) in TFA (0.2 ml) and MeOH (2ml) was stirred at room temperature for 24 hours, then evaporated tilldryness. The residue (0.05 g) was purified by column chromatography overamino coated silica gel (25-40 μm) (eluent: DCM/MeOH/water 85/15/1). Thepure fractions were collected and the solvent was evaporated, yielding0.022 g (56%) of compound 4, melting point 70° C.

Example B5 Preparation of Compound 5

Triethylamine (0.0027 mol) was added at room temperature to a solutionof intermediate 13 (0.0006 mol), 1,2-benzenediamine (0.0016 mol) andbenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate(0.0014 mol) in DCM (5 ml) and THF (5 ml). The mixture was stirred atroom temperature for 72 hours, poured out into water and extracted withDCM. The organic layer was separated, dried (MgSO₄), filtered and thesolvent was evaporated. The residue (0.8 g) was purified by columnchromatography over kromasil (3.5 μm) (eluent: DCM/MeOH/NH₄OH 97/3/0.3to 92/8/0.3). The pure fractions were collected and the solvent wasevaporated, yielding 0.014 g (4%) of compound 5, melting point 90° C.

Table F-1 lists the compounds that were prepared according to one of theabove Examples.

TABLE F-1

C. Pharmacological Example

The in vitro assay for inhibition of histone deacetylase (see exampleC.1) measures the inhibition of HDAC enzymatic activity obtained withthe compounds of formula (I).

Cellular activity of the compounds of formula (I) was determined onA2780 tumour cells using a calorimetric assay for cell toxicity orsurvival (Mosmann Tim, Journal of Immunological Methods 65: 55-63,1983)(see example C.2).

The solubility of a compound measures the ability of a compound to stayin solution. In the first method, the ability of a compound to stay inaqueous solution upon dilution (see example C.3.a) is measured.DMSO-stock solutions are diluted with a single aqueous buffer solvent indifferent consecutive steps. Mixtures are then scanned in the BD GentestSolubility Scanner for the occurrence of precipitation. In the secondmethod the solubility of a compound at different pH's can be measuredwith the use of a chemiluminescent nitrogen detector (see exampleC.3.b).

A drug's permeability expresses its ability to move from one medium intoor through another. Specifically its ability to move through theintestinal membrane into the blood stream and/or from the blood streaminto the target. Permeability (see example C.4) can be measured throughthe formation of a filter-immobilized artificial membrane phospholipidbilayer. In the filter-immobilized artificial membrane assay, a“sandwich” is formed with a 96-well microtitre plate and a 96-wellfilter plate, such that each composite well is divided into two chamberswith a donor solution at the bottom and an acceptor solution at the top,separated by a 125 μm micro-filter disc (0.45 μm pores), coated with 2%(wt/v) dodecane solution of dioleoylphosphatidyl-choline, underconditions that multi-lamellar bilayers form inside the filter channelswhen the system contacts an aqueous buffer solution. The permeability ofcompounds through this artificial membrane is measured in cm/s. Thepurpose is to look for the permeation of the drugs through a parallelartificial membrane at 2 different pH's: 4.0 and 7.4. Compound detectionis done with UV-spectrometry at optimal wavelength between 250 and 500nm.

Metabolism of drugs means that a lipid-soluble xenobiotic or endobioticcompound is enzymatically transformed into (a) polar, water-soluble, andexcretable metabolite(s). The major organ for drug metabolism is theliver. The metabolic products are often less active than the parent drugor inactive. However, some metabolites may have enhanced activity ortoxic effects. Thus drug metabolism may include both “detoxication” and“toxication” processes. One of the major enzyme systems that determinethe organism's capability of dealing with drugs and chemicals isrepresented by the cytochrome P450 monooxygenases, which are NADPHdependent enzymes. Metabolic stability of compounds can be determined invitro with the use of subcellular human tissue (see example C.5.). Heremetabolic stability of the compounds is expressed as % of drugmetabolised after 15 minutes incubation of these compounds withmicrosomes. Quantitation of the compounds was determined by LC-MSanalysis.

It has been shown that a wide variety of anti-tumoral agents activatethe p21 protein, including DNA damaging agents and histone deacetylaseinhibitors. DNA damaging agents activate the p21 gene through the tumoursuppressor p53, while histone deacetylase inhibitors transcriptionallyactivates the p21 gene via the transcription factor Sp1. Thus, DNAdamaging agents activate the p21 promoter through the p53 responsiveelement while histone deacetylase inhibitors activate the p21 promoterthrough sp1 sites (located at the −60 bp to +40 bp region relative tothe TATA box) both leading to increased expression of the p21 protein.When the p21 promoter in a cells consists of a p21 1300 bp promoterfragment that does not comprise the p53 responsive elements it isaccordingly non-responsive to DNA damaging agents. The capacity ofcompounds to induce p21 can be evaluated by testing the capacity ofcompounds to induce p21 as the consequence of HDAC inhibition at thecellular level. The cells can be stably transfected with an expressionvector containing a p21 1300 bp promoter fragment that does not comprisethe p53 responsive elements and wherein an increase of a reporter geneexpression, compared to the control levels, identifies the compound ashaving p21 induction capacity. The reporter gene is a fluorescentprotein and the expression of the reporter gene is measured as theamount of fluorescent light emitted (see example C.6.a).

The last method is an in vivo method wherein mice are used for screeningthe pharmaceutical activity of a compound. The above described stablytransformed tumour cells can be administered to mice in an amountsufficient to effect production of a tumour. After the tumour cells hadsufficient time to form a tumour, a potentially active compound can beadministered to the animals and the effect of said compound on thetumour cells is evaluated by measuring the expression of the reportergene. Incubation with pharmaceutical active compounds will result in anincrease of reporter gene expression compared to the control levels (seeexample C.6.b.)

Specific HDAC inhibitors should not inhibit other enzymes like theabundant CYP P450 proteins. The CYP P450 (E. coli expressed) proteins3A4, 2D6 en 2C9 convert their specific substrates into a fluorescentmolecule. The CYP3A4 protein converts 7-benzyloxy-trifluoromethylcoumarin (BFC) into 7-hydroxy-trifluoromethyl coumarin. The CYP2D6protein converts3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC)into 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarinhydrochloride and the CYP2C9 protein converts7-Methoxy-4-trifluoromethyl coumarin (MFC) into7-hydroxy-trifluoromethyl coumarin. Compounds inhibiting the enzymaticreaction will result in a decrease of fluorescent signal (see exampleC.7).

Example C.1 In Vitro Assay for Inhibition of Histone Deacetylase

The HDAC Fluorescent Activity Assay/Drug Discovery Kit of Biomol (cat.No: AK-500-0001) was used. The HDAC Fluorescent Activity Assay is basedon the Fluor de Lys (Fluorogenic Histone deAcetylase Lysyl) substrateand developer combination. The Fluor de Lys substrate, comprises anacetylated lysine side chain. Deacetylation of the substrate sensitizesthe substrate so that, in the second step, treatment with the Fluor deLys developer produces a fluorophore.

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg/ml with75 μM of substrate. The Fluor de Lys substrate was added in a buffercontaining 25 mM Tris, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl₂.6H₂O at pH7.4. After 30 min, 1 volume of the developer was added. The fluorophorewas excited with 355 nm light and the emitted light (450 nm) was bedetected on a fluorometric plate reader. For each experiment, controls(containing HeLa nuclear extract and buffer), a blank incubation(containing buffer but no HeLa nuclear extract) and samples (containingcompound dissolved in DMSO and further diluted in buffer and HeLanuclear extract) were run in parallel. In first instance, compounds weretested at a concentration of 10⁻⁵M. When the compounds showed activityat 10⁻⁵M, a concentration-response curve was made wherein the compoundswere tested at concentrations between 10⁻⁵M and 10⁻⁹M. All sample weretested 4 times. In each test the blank value was subtracted from boththe control and the sample values. The control sample represented 100%of substrate deacylation. For each sample the fluorescence was expressedas a percentage of the mean value of the controls. When appropriateIC₅₀-values (concentration of the drug, needed to reduce the amount ofmetabolites to 50% of the control) were computed using probit analysisfor graded data. Herein the effects of test compounds are expressed aspIC₅₀ (the negative log value of the IC₅₀-value) (see Table F-2).

Example C.2 Determination of Antiproliferative Activity on A2780 Cells

All compounds tested were dissolved in DMSO and further dilutions weremade in culture medium. Final DMSO concentrations never exceeded 0.1%(v/v) in cell proliferation assays. Controls contained A2780 cells andDMSO without compound and blanks contained DMSO but no cells. MTT wasdissolved at 5 mg/ml in PBS. A glycine buffer comprised of 0.1 M glycineand 0.1 M NaCl buffered to pH 10.5 with NaOH (1 N) was prepared (allreagents were from Merck).

The human A2780 ovarian carcinoma cells (a kind gift from Dr. T. C.Hamilton [Fox Chase Cancer Centre, Pa., USA]) were cultured in RPMI 1640medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicin and 10%fetal calf serum. Cells were routinely kept as monolayer cultures at 37°C. in a humidified 5% CO₂ atmosphere. Cells were passaged once a weekusing a trypsin/EDTA solution at a split ratio of 1:40. All media andsupplements were obtained from Life Technologies. Cells were free ofmycoplasma contamination as determined using the Gen-Probe MycoplasmaTissue Culture kit (supplier: BioMérieux).

Cells were seeded in NUNC™ 96-well culture plates (Supplier: LifeTechnologies) and allowed to adhere to the plastic overnight. Densitiesused for plating were 1500 cells per well in a total volume of 200 μlmedium. After cell adhesion to the plates, medium was changed and drugsand/or solvents were added to a final volume of 200 μl. Following fourdays of incubation, medium was replaced by 200 μl fresh medium and celldensity and viability was assessed using an MTT-based assay. To eachwell, 25 μl MTT solution was added and the cells were further incubatedfor 2 hours at 37° C. The medium was then carefully aspirated and theblue MTT-formazan product was solubilized by addition of 25 μl glycinebuffer followed by 100 μl of DMSO. The microtest plates were shaken for10 min on a microplate shaker and the absorbance at 540 nm was measuredusing an Emax 96-well spectrophotometer (Supplier: Sopachem). Within anexperiment, the results for each experimental condition are the mean of3 replicate wells. For initial screening purposes, compounds were testedat a single fixed concentration of 10⁻⁶ M. For active compounds, theexperiments were repeated to establish full concentration-responsecurves. For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was subtracted from all control and sample values. For eachsample, the mean value for cell growth (in absorbance units) wasexpressed as a percentage of the mean value for cell growth of thecontrol. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce cell growth to 50% of the control) were computed usingprobit analysis for graded data (Finney, D. J., Probit Analyses, 2^(nd)Ed. Chapter 10, Graded Responses, Cambridge University Press, Cambridge1962). Herein the effects of test compounds are expressed as pIC₅₀ (thenegative log value of the IC₅₀-value)(see Table F-2).

Example C.3 Solubility/Stability C.3.a. Kinetic Solubility in AqueousMedia

DMSO-stock solutions from 5000-9.8 μM (½ dilutions) are made in DMSO ina 96 well stock solution plate (200 μl per well). After each dilutionthe samples are mixed. Aliquots of these DMSO solutions (2 μl) are thentransferred into 2 other 96 well buffer plates, containing 200 μl perwell aqueous buffer. Each of the buffer plates contains either aqueousbuffer pH 7.4 or aqueous buffer pH 4.0. After the last dilution thebuffer plates are mixed and the samples are stabilized at roomtemperature for ½ hour. Dilution is done in duplicate for each compoundto exclude occasional errors. Mixtures are then scanned in the BDGentest Solubility Scanner for the occurrence of precipitation. Based onthe absence/presence of precipitate in the mixtures the kineticsolubility is calculated by interpolation. Ranking is performed into the3 classes.

Compounds with high solubility obtained a score of 3 and have asolubility higher than or equal to 50 μM. Compounds with mediumsolubility obtained a score of 2 and have a solubility higher than 10 μMand lower than 50 μM. Compounds with low solubility obtained a score of1 and for these compounds solubility is lower than or equal to 10 μM.(see Table F-2).

C.3.b. Solubility

The solubility of a compound, at different pH's, can also be measuredwith the use of a chemiluminescent nitrogen detector (see Table F-2).

Example C.4 Parallel Artificial Membrane Permeability Analysis

The stock samples (aliquots of 10 μl of a stock solution of 5 mM in 100%DMSO) were diluted in a deep-well or Pre-mix plate containing 2 ml of anaqueous buffer system pH 4 or pH 7.4 (PSR4System Solution Concentrate(pION)). Before samples were added to the reference plate, 150 μl ofbuffer was added to wells and a blank UV-measurement was performed.Thereafter the buffer was discarded and the plate was used as referenceplate. All measurements were done in UV-resistant plates (supplier:Costar or Greiner).

After the blank measurement of the reference plate, 150 μl of thediluted samples was added to the reference plate and 200 μl of thediluted samples was added to donorplate 1. An acceptor filter plate 1(supplier: Millipore, type:MAIP N45) was coated with 4 μl of theartificial membrane-forming solution(1,2-Dioleoyl-sn-Glycer-3-Phosphocholine in Dodecane containing 0.1%2,6-Di-tert-butyl-4-methylphenol and placed on top of donor plate 1 toform a “sandwich”. Buffer (200 μl) was dispensed into the acceptor wellson the top. The sandwich was covered with a lid and stored for 18 h atroom temperature in the dark.

A blank measurement of acceptor plate 2 was performed through theaddition of 150 μl of buffer to the wells, followed by anUV-measurement. After the blank measurement of acceptor plate 2 thebuffer was discarded and 150 μl of acceptor solution was transferredfrom the acceptor filter plate 1 to the acceptor plate 2. Then theacceptor filter plate 1 was removed form the sandwich. After the blankmeasurement of donor plate 2 (see above), 150 μl of the donor solutionwas transferred from donor plate 1 to donor plate 2. The UV spectra ofthe donor plate 2, acceptor plate 2 and reference plate wells werescanned (with a SpectraMAX 190). All the spectra were processed tocalculate permeability with the PSR4p Command Software. All compoundswere measured in triplo. Carbamazepine, griseofulvin, acycloguanisine,atenolol, furosemide, and chlorothiazide were used as standards in eachexperiment. Compounds were ranked in 3 categories as having a lowpermeability (mean effect <0.5×10⁻⁶ cm/s; score 1), a mediumpermeability (1×10⁻⁶ cm/s >mean effect ≧0.5×10⁻⁶ cm/s; score 2) or ahigh permeability (≧1×10⁻⁶ cm/s; score 3).

Example C.5 Metabolic Stability

Sub-cellular tissue preparations were made according to Gorrod et al.(Xenobiotica 5: 453-462, 1975) by centrifugal separation aftermechanical homogenization of tissue. Liver tissue was rinsed in ice-cold0.1 M Tris-HCl (pH 7.4) buffer to wash excess blood. Tissue was thenblotted dry, weighed and chopped coarsely using surgical scissors. Thetissue pieces were homogenized in 3 volumes of ice-cold 0.1 M phosphatebuffer (pH 7.4) using either a Potter-S (Braun, Italy) equipped with aTeflon pestle or a Sorvall Omni-Mix homogeniser, for 7×10 sec. In bothcases, the vessel was kept in/on ice during the homogenization process.

Tissue homogenates were centrifuged at 9000×g for 20 minutes at 4° C.using a Sorvall centrifuge or Beckman Ultracentrifuge. The resultingsupernatant was stored at −80° C. and is designated ‘S9’.

The S9 fraction can be further centrifuged at 100.000×g for 60 minutes(4° C.) using a Beckman ultracentrifuge. The resulting supernatant wascarefully aspirated, aliquoted and designated ‘cytosol’. The pellet wasre-suspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1ml per 0.5 g original tissue weight and designated ‘microsomes’.

All sub-cellular fractions were aliquoted, immediately frozen in liquidnitrogen and stored at −80° C. until use.

For the samples to be tested, the incubation mixture contained PBS(0.1M), compound (5 μM), microsomes (1 mg/ml) and a NADPH-generatingsystem (0.8 mM glucose-6-phosphate, 0.8 mM magnesium chloride and 0.8Units of glucose-6-phosphate dehydrogenase). Control samples containedthe same material but the microsomes were replaced by heat inactivated(10 min at 95 degrees Celsius) microsomes. Recovery of the compounds inthe control samples was always 100%.

The mixtures were preincubated for 5 min at 37 degrees Celsius. Thereaction was started at timepoint zero (t=0) by addition of 0.8 mM NADPand the samples were incubated for 15 min (t=15). The reaction wasterminated by the addition of 2 volumes of DMSO. Then the samples werecentrifuged for 10 min at 900×g and the supernatants were stored at roomtemperature for no longer as 24 h before analysis. All incubations wereperformed in duplo. Analysis of the supernatants was performed withLC-MS analysis. Elution of the samples was performed on a Xterra MS C18(50×4.6 mm, 5 μm, Waters, US). An Alliance 2790 (Supplier: Waters, US)HPLC system was used. Elution was with buffer A (25 mM ammoniumacetate(pH 5.2) in H₂O/acetonitrile (95/5)), solvent B being acetonitrile andsolvent C methanol at a flow rate of 2.4 ml/min. The gradient employedwas increasing the organic phase concentration from 0% over 50% B and50% C in 5 min up to 100% B in 1 min in a linear fashion and organicphase concentration was kept stationary for an additional 1.5 min. Totalinjection volume of the samples was 25 μl.

A Quattro (supplier: Micromass, Manchester, UK) triple quadrupole massspectrometer fitted with and ESI source was used as detector. The sourceand the desolvation temperature were set at 120 and 350° C. respectivelyand nitrogen was used as nebuliser and drying gas. Data were acquired inpositive scan mode (single ion reaction). Cone voltage was set at 10 Vand the dwell time was 1 sec.

Metabolic stability was expressed as % metabolism of the compound after15 min of incubation in the presence of active microsomes

$\left( {E({act})} \right)\left( {{\% \mspace{14mu} {metabolism}} = {{100\%} - {\left( {\left( \frac{{{Total}\mspace{14mu} {Ion}\mspace{14mu} {Current}\mspace{14mu} ({TIC})\mspace{14mu} {of}\mspace{14mu} {E({act})}\mspace{14mu} {at}\mspace{14mu} t} = 15}{{{TIC}\mspace{14mu} {of}\mspace{14mu} {E({act})}\mspace{14mu} {at}\mspace{14mu} t} = 0} \right) \times 100} \right).}}} \right.$

Compounds that had a percentage metabolism less than 20% were defined ashighly metabolic stable. Compound that had a metabolism between 20 and70% were defined as intermediately stable and compounds that showed apercentage metabolism higher than 70 were defined as low metabolicstable. Three reference compounds were always included whenever ametabolic stability screening was performed. Verapamil was included as acompound with low metabolic stability (% metabolism=73%). Cisapride wasincluded as a compound with medium metabolic stability (% metabolism45%) and propanol was included as a compound with intermediate to highmetabolic stability (25% metabolism). These reference compounds wereused to validate the metabolic stability assay. Compounds 2 and 3 weretested and were respectively found highly metabolically stable andintermediately stable.

Example C.6 p21 Induction Capacity Example C.6.a Cellular Method

A2780 cells (ATCC) were cultivated in RPMI 1640 medium supplemented with10% FCS, 2 mM L-glutamine and gentamycine at 37° C. in a humidifiedincubator with 5% CO₂. All cell culture solutions are provided byGibco-BRL (Gaithersburg, Md.). Other materials are provided by Nunc.

Genomic DNA was extracted from proliferating A2780 cells and used astemplate for nested PCR isolation of the p21 promoter. The firstamplification was performed for 20 cycles at an annealing temperature of55° C. using the oligonucleotide pair GAGGGCGCGGTGCTTGG andTGCCGCCGCTCTCTCACC with the genomic DNA as template. The resulting 4.5kb fragment containing the −4551 to +88 fragment relative to the TATAbox was re-amplified with the oligonucleotidesTCGGGTACCGAGGGCGCGGTGCTTGG and ATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycleswith annealing at 88° C. resulting in a 4.5 kb fragment and subsequentlywith the oligonucleotide pair TCGGGTACCGGTAGATGGGAGCGGATAGACACATC andATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycles with annealing at 88° C.resulting in a 1.3 kb fragment containing the −1300 to +88 fragmentrelative to the TATA box. The restriction sites XhoI and KpnI present inthe oligonucleotides (underlined sequence) were used for subcloning.

The luciferase reporter was removed from the pGL3-basic and replaced bythe ZsGreen reporter (from the pZsGreen1-N1 plasmid) at KpnI and XbaIrestriction sites. pGL3-basic-ZsGreen-1300 was constructed via insertionof the above mentioned 1.3 kb fragment of the human p21 promoter regioninto pGL3-basic-ZsGreen at the XhoI and KpnI sites. All restrictionenzymes are provided by Boehringer Manheim (Germany). A2780 cells wereplated into a 6-well plate at a density of 2×10⁵ cells, incubated for 24hours, and transfected with 2 ug of pGL3-basic-ZsGreen-1300 and 0.2 ugof pSV2neo vector by using Lipofectamine 2000 (Invitrogen, Brussels,Belgium) as described by manufacturer. The transfected cells wereselected for 10 days with G418 (Gibco-BRL, Gaithersburg, Md.) and singlecell suspensions were grown. After three weeks, single clones wereobtained.

The A2780 selected clones were expanded and seeded at 10000 cells perwell into 96-well plates. 24 hours after seeding, the cells were treatedfor an additional 24 hours with compounds (affecting sp1 sites in theproximal p21 promoter region). Subsequently, cells were fixed with 4%PFA for 30′ and counterstained with Hoechst dye. The p21 promoteractivation leading to ZsGreen production and thus fluorescence, wasmonitored by the Ascent Fluoroskan (Thermo Labsystems, Brussels,Belgium).

For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was subtracted from all control and sample values. For eachsample, the value for p21 induction was expressed as the percentage ofthe value for p21 present in the control. Percentage induction higherthan 130% was defined as significant induction.

Compounds 1, 2 and 3 were tested and showed significant induction at aconcentration of 100 nM.

Example C.6.b. In Vivo Method

A selected clone was injected subcutaneous (10⁷ cells/200 μl) into theflank of nude mice and a calliper measurable tumour was obtained after12 days. From day 12 on, animals were dosed, orally or intraveinally,daily during 6 days with solvent and 20-40 mpk compound (4-10 animalseach). Tumours were evaluated for fluorescence by the in-house developedAutomated Whole Body Imaging System (Fluorescent stereomicroscope typeOlympus® SZX12 equipped with a GFP filter and coupled to a CCD cameratype JAI® CV-M90 controlled by a software package based on the IMAQVision Software from National Instruments®).

Compound 2 was tested and showed increased fluorescence as compared tocontrol.

Example C.7 P450 Inhibiting Capacity

All compounds tested were dissolved in DMSO (5 mM) and a furtherdilution to 5 10⁻⁴ M was made in acetonitrile. Further dilutions weremade in assay buffer (0.1M NaK phosphate buffer pH 7.4) and the finalsolvent concentration was never higher than 2 The assay for the CYP3A4protein comprises per well 15 μmol P450/mg protein (in 0.01MNaKphosphate buffer+1.15% KCl), an NADPH generating system (3.3 mMGlucose-6-phosphate, 0.4 U/ml Glucose-6-phosphate dehydrogenase, 1.3 mMNADP and 3.3 mM MgCl₂.6H₂O in assay buffer) and compound in a totalassay volume of 100 μl. After a 5 min pre-incubation at 37° C. theenzymatic reaction was started with the addition of 150 μM of thefluorescent probe substrate BFC in assay buffer. After an incubation of30 minutes at room temperature the reaction was terminated afteraddition of 2 volumes of acetonitrile. Fluorescent determinations werecarried out at an excitation wavelength of 405 nm and an emissionwavelength of 535 nm. Ketoconazole (IC₅₀-value=3×10⁻⁸M) was included asreference compound in this experiment. The assay for the CYP2D6 proteincomprises per well 6 pmol P450/mg protein (in 0.01M NaKphosphatebuffer+1.15% KCl), an NADPH generating system (0.41 mMGlucose-6-phosphate, 0.4 U/ml Glucose-6-phosphate dehydrogenase, 0.0082mM NADP and 0.41 mM MgCl₂.6H₂O in assay buffer) and compound in a totalassay volume of 100 μl. After a 5 min pre-incubation at 37° C. theenzymatic reaction was started with the addition of 3 μM of thefluorescent probe substrate AMMC in assay buffer. After an incubation of45 minutes at room temperature the reaction was terminated afteraddition of 2 volumes of acetonitrile. Fluorescent determinations werecarried out at an excitation wavelength of 405 nm and an emissionwavelength of 460 nm. Quinidine (IC₅₀-value <5×10⁻⁸ M) was included asreference compound in this experiment.

The assay for the CYP2C9 protein comprises per well 15 pmol P450/mgprotein (in 0.01M NaKphosphate buffer+1.15% KCl), an NADPH generatingsystem (3.3 mM Glucose-6-phosphate, 0.4 U/ml Glucose-6-phosphatedehydrogenase, 1.3 mM NADP and 3.3 mM MgCl₂.6H₂O in assay buffer) andcompound in a total assay volume of 100 μl. After a 5 min pre-incubationat 37° C. the enzymatic reaction was started with the addition of 200 μMof the fluorescent probe substrate MFC in assay buffer. After anincubation of 30 minutes at room temperature the reaction was terminatedafter addition of 2 volumes of acetonitrile. Fluorescent determinationswere carried out at an excitation wavelength of 405 nm and an emissionwavelength of 535 nm. Sulfaphenazole (IC₅₀-value=6.8×10⁻⁷ M) wasincluded as reference compound in this experiment.

For initial screening purposes, compounds were tested at a single fixedconcentration of 1×10⁻⁵ M. For active compounds, the experiments wererepeated to establish full concentration-response curves. For eachexperiment, controls (containing no drug) and a blank incubation(containing no enzyme or drugs) were run in parallel. All compounds wereassayed in quadruplicate. The blank value was subtracted from allcontrol and sample values. For each sample, the mean value of P450activity of the sample (in relative fluorescence units) was expressed asa percentage of the mean value of P450 activity of the control.Percentage inhibition was expressed as 100% minus the mean value of P450activity of the sample. When appropriate, IC₅₀-values (concentration ofthe drug, needed to reduce P450 activity to 50% of the control) werecalculated.

TABLE F-2 lists the results of the compounds that were tested accordingto example C.1, C.2, C.3.a. and C.3.b. Solubility Solubility EnzymaticCellular C.3.a. C.3.b. activity activity pH = pH = Co. No. pIC50 C.1.pIC50 C.2. 7.4 μm 2.3 mg/ml 1 9.0 7.5 7.8 2 8.9 7.3 9.4 1.35 3 8.3 7.515.6

D. Composition Example Film-Coated Tablets Preparation of Tablet Core

A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 gstarch is mixed well and thereafter humidified with a solution of 5 gsodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 mlof water. The wet powder mixture is sieved, dried and sieved again. Thenthere is added 100 g microcrystalline cellulose and 15 g hydrogenatedvegetable oil. The whole is mixed well and compressed into tablets,giving 10.000 tablets, each comprising 10 mg of a compound of formula(I).

Coating

To a solution of 10 g methyl cellulose in 75 ml of denatured ethanolthere is added a solution of 5 g of ethyl cellulose in 150 ml ofdichloromethane. Then there are added 75 ml of dichloromethane and 2.5ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten anddissolved in 75 ml of dichloromethane. The latter solution is added tothe former and then there are added 2.5 g of magnesium octadecanoate, 5g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspensionand the whole is homogenated. The tablet cores are coated with the thusobtained mixture in a coating apparatus.

1. A compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein n is 0 or 1 andwhen n is 0 than a direct bond is intended; m is 0, 1 or 2 and when n is0 than a direct bond is intended; p is 0 or 1 and when n is 0 than adirect bond is intended; each X is independently N or CH; each Y isindependently O, NH, N—C₁₋₆alkyl, CH or CH₂ and when Y is CH then thesubstituent is attached to the Y atom of the ring structure; R¹ ishydroxy or a radical of formula (a-1)

wherein R⁹ is hydroxy or —NH₂; R¹⁰ is hydrogen, thienyl, furanyl orphenyl and each thienyl, furanyl or phenyl can optionally be substitutedwith halo, amino, nitro, cyano, hydroxy, phenyl, C₁₋₆alkyl,(diC₁₋₆alkyl)amino, C₁₋₆alkyloxy, phenylC₁₋₆alkyloxy, hydroxyC₁₋₆alkyl,C₁₋₆alkyloxycarbonyl, hydroxycarbonyl, C₁₋₆alkylcarbonyl,polyhaloC₁₋₆alkyloxy, polyhaloC₁₋₆alkyl, C₁₋₆alkylsulfonyl,hydroxycarbonylC₁₋₆alkyl, C₁₋₆alkylcarbonylamino, aminosulfonyl,aminosulfonylC₁₋₆alkyl, isoxazolyl, aminocarbonyl, phenylC₂₋₆alkenyl,phenylC₃₋₆alkynyl or pyridinylC₃₋₆alkynyl; R⁶, R⁷ and R⁸ are eachindependently hydrogen, —NH₂, nitro, furanyl, halo, C₁₋₆alkyl,C₁₋₆alkyloxy, trifluoromethyl, thienyl, phenyl, C₁₋₆alkylcarbonylamino,aminocarbonylC₁₋₆alkyl or —C≡C—CH₂—R¹¹; wherein R¹″ is hydrogen,C₁₋₆alkyl, hydroxy, amino or C₁₋₆alkyloxy; R² is C₁₋₆alkyl,C₃₋₇cycloalkyl, C₁₋₆alkylaminocarbonyl or C₁₋₆alkyloxycarbonyl; R³ ishydrogen, C₁₋₆alkyl, C₃₋₇cycloalkyl, hydroxyC₁₋₆alkyl,C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxycarbonyl or C₁₋₆alkylaminocarbonyl;or R² and R³ can be bridged (i.e. forming a cyclic ring system) with amethylene, ethylene or propylene bridge; R⁴ is hydrogen, C₁₋₆alkyl,—C(═O)—CHR¹²R¹³ or —S(═O)₂—N(CH₃)₂; wherein each R¹² and R¹³ isindependently hydrogen, amino, C₁₋₆alkyl or aminoC₁₋₆alkyl; and R⁵ ishydrogen, hydroxy, amino, halo, C₁₋₆alkyl, polyhaloC₁₋₆alkyl,C₁₋₆alkyloxycarbonyl, hydroxycarbonyl, C₁₋₆alkylcarbonylamino,C₁₋₆alkyloxy, or mono- or di(C₁₋₆alkyl)amino.
 2. A compound according toclaim 1 wherein n is 1; p is 0; each Y is CH; R¹⁰ is hydrogen; R⁶, R⁷and R⁸ are each independently hydrogen, R² is C₁₋₆alkyl and R³ ishydrogen or R² and R³ can be bridged with a methylene, ethylene orpropylene bridge; R⁴ is C₁₋₆alkyl; and R⁵ is hydrogen.
 3. A compoundaccording to claim 1 wherein n is 1; m is 1 or 2; p is 0; each X is N;each Y is CH; R² and R³ are bridged with a methylene, ethylene orpropylene bridge; R⁴ is C₁₋₆alkyl; and R⁵ is hydrogen.
 4. A compoundthat is selected from the group consisting of: compound No. 2 andcompound No.
 3.


5. A pharmaceutical composition comprising pharmaceutically acceptablecarriers and as an active ingredient a therapeutically effective amountof a compound as claimed in claim
 1. 6. (canceled)
 7. (canceled)
 8. Amethod of treating in a subject proliferative disease, comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 9. A combination of an anti-canceragent and a HDAC inhibitor as claimed in claim
 1. 10. A process forpreparing a compound as claimed in claim 1, said process comprising thesteps of: a) reacting an intermediate of formula (II) with anappropriate acid yielding a hydroxamic acid of formula (I), wherein R¹is hydroxy, herein referred to as compounds of formula (I-a)

b) reacting an intermediate of formula (III) wherein M representshydrogen or sodium or lithium or an alkali metal cation, with anintermediate of formula (IV) in the presence of appropriate reagents andin a suitable solvent, with the formation of a compound of formula (I),wherein R¹ is a radical of formula (a-1) and R⁹ is —NH₂, herein referredto as compounds of formula (I-b),

c) reacting an intermediate of formula (V) with an appropriate acid in asuitable solvent, with the formation of a compound of formula (I-b), or

d) reacting an intermediate of formula (VI), wherein TBDMS meanstert-butyl(dimethyl)silanyl, with tetrabutylammonium fluoride in asuitable solvent, with the formation of a compound of formula (I),wherein R¹ is a radical of formula (a-1) and R⁹ is hydroxy, hereinreferred to as compounds of formula (I-c).


11. A compound of formula (A):

in which Q is C₁₋₂alkyloxycarbonyl, hydroxycarbonyl ortetrahydropyranyloxyaminocarbonyl and n, m, p, X, Y, Z, R², R³, R⁴, andR⁵ are as defined for formula (I) in claim 1, and the N-oxide forms, thepharmaceutically acceptable addition salts and the stereo-chemicallyisomeric forms thereof.
 12. A process for preparing a compound asclaimed in claim 11, said process comprising the steps of: a) reactingan intermediate of formula (III-a) with an intermediate of formula (VII)to form a compound of formula (A) in which Q istetrahydropyranyloxyaminocarbonyl, represented by formula (II):

b) reacting the intermediate of formula (X) with an appropriate acidicsolution, to form a compound of formula (A), wherein Q ishydroxycarbonyl herein referred to as a compound of formula (III),

c) reacting the intermediate of formula (XI) with an intermediate offormula (XII), to form a compound of formula (A) wherein Q isC₁₋₂alkyloxycarbonyl and R³ is hydrogen herein referred to as compoundsof formula (X-a), or

d) reacting the intermediate of formula (XIII) with an intermediate offormula (XIV), to form a compound of formula (A) wherein Q isC₁₋₂alkyloxycarbonyl and R² and R³ are bridged, the dotted linerepresents a methylene, ethylene or propylene bridge and n is 1, hereinreferred to as intermediates of formula (X-b).


13. A compound according to claim 2 wherein n is 1; m is 1 or 2; p is 0;each X is N; each Y is CH; R² and R³ are bridged with a methylene,ethylene or propylene bridge; R⁴ is C₁₋₆alkyl; and R⁵ is hydrogen.
 14. Apharmaceutical composition comprising pharmaceutically acceptablecarriers and as an active ingredient a therapeutically effective amountof a compound as claimed in claim
 2. 15. A pharmaceutical compositioncomprising pharmaceutically acceptable carriers and as an activeingredient a therapeutically effective amount of a compound as claimedin claim
 3. 16. A pharmaceutical composition comprising pharmaceuticallyacceptable carriers and as an active ingredient a therapeuticallyeffective amount of a compound as claimed in claim 4.